Monoclonal Antibodies Against Alpha-Synuclein Fibrils

ABSTRACT

The present disclosure provides monoclonal antibodies that bind α-Synuclein. In certain aspects, the antibodies preferentially bind to α-Synuclein fibrils over α-Synuclein monomer. In other aspects, the invention comprises a method of treating α-Synucleopathic disease in a subject, comprising administering any of the antibodies of the invention to the subject. In yet other aspects, the invention comprises methods of detecting α-Synuclein fibrils using any of the antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of, and claims priority to,U.S. application Ser. No. 16/609,885, filed Oct. 31, 2019, now allowed,which is a 35 U.S.C. § 371 national phase application from, and priorityto, International Application No. PCT/US2018/030436, filed May 1, 2018and published under PCT Article 21(2) in English, which claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.62/492,663, filed May 1, 2017, all of which applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberNS053488 awarded by National Institute of Neurological Disorders andStroke (NIH) and grant number T32-AG000255 awarded by NationalInstitutes of Aging (NIH). The government has certain rights in theinvention.

SEQUENCE LISTING

The ASCII text file named “046483-7153US2_Seq Listing_ST25.txt” createdon Jan. 6, 2022, comprising 13,681 bytes, is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The fibrillization of α-Synuclein (α-Syn) is considered to play a keyrole in the pathogenesis of several neurodegenerative diseases,including Parkinson's disease (PD), dementia with Lewy bodies (DLB), andmultiple system atrophy (MSA). Genetic studies show various pointmutations in α-Syn (A53T, A30P, E46K, H50Q or G51D) and multiplicationof the α-Syn gene SNCA lead to familial PD. Cell culture and animalmodel studies indicate that enhanced oligomerization and aggregation ofα-Syn is associated with increased cytotoxicity, while α-Syn oligomershave been shown to be significantly elevated in the brain lysates andcerebrospinal fluid of PD and DLB patients. The significance of α-Synfibrillization in the pathobiology of synucleinopathies has beenreinforced by recent data demonstrating that preformed recombinant α-Synfibrils can seed the aggregation of endogenous α-Syn both in a varietyof neurons cultured from wild-type mice, and in vivo in transgenic andwildtype mice, even from the peripheral to the central nervous system.

Given the central role of α-Syn in the pathogenesis ofsynucleinopathies, the characterization of its pathology in the brainhas relied heavily on the use of α-Syn antibodies, the vast majority ofwhich recognize both the monomeric and the aggregated forms of α-Syn.Hence, there is a need for developing conformation-specific antibodiesthat can unveil underappreciated α-Syn neuropathology or even revealnovel neuropathological features of PD and related disorders.Furthermore, taking into account that passive immunotherapy againstα-Syn has emerged as a very promising strategy for modifying PD andrelated synucleinopathies, the generation of high-affinity and/orconformation-specific antibodies that recognize α-Syn fibrils may proveto be a useful tool for the treatment of these diseases. This disclosureaddresses and meets those needs.

SUMMARY

Provided is an isolated monoclonal antibody, or fragment thereof, whichrecognizes a conformational epitope comprising amino acids 110-120 ofα-Synuclein (α-Syn), and wherein affinity of the antibody for α-Synfibrils is higher than for α-Syn monomers. In some embodiments, theantibody binds at least about 30 times more tightly to α-Syn fibrilsthan to α-Syn monomers.

Provided is an isolated monoclonal antibody, or fragment thereof, whichrecognizes a conformational epitope comprising amino acids 120-130 ofα-Synuclein (α-Syn), and wherein affinity of the antibody for α-Synfibrils is similar to that for α-Syn monomers. In some embodiments, theantibody binds to α-Syn monomers with an affinity equal to or tighterthan about 10⁻⁹ M.

Provided is an isolated monoclonal antibody comprising a light chainvariable region (VL) and a heavy chain variable region (VH), wherein theVL comprises a CDR1 region comprising the amino acid sequence of SEQ IDNOs: 1, 11 or 21; a CDR2 region comprising the amino acid sequence ofSEQ ID NOs: 2, 12 or 22; and a CDR3 region comprising the amino acidsequence of SEQ ID NOs: 3, 13 or 23, and wherein the VH comprises a CDR1region comprising the amino acid sequence of SEQ ID NOs: 6, 16 or 26; aCDR2 region comprising the amino acid sequence of SEQ ID NOs: 7, 17 or27; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs:8, 18 or 28. In some embodiments, the VL comprises a CDR1 regioncomprising the amino acid sequence of SEQ ID NOs: 1 or 11; a CDR2 regioncomprising the amino acid sequence of SEQ ID NOs: 2 or 12; and a CDR3region comprising the amino acid sequence of SEQ ID NOs: 3 or 13, andwherein the VH comprises a CDR1 region comprising the amino acidsequence of SEQ ID NOs: 6 or 16; a CDR2 region comprising the amino acidsequence of SEQ ID NOs: 7 or 17; and a CDR3 region comprising the aminoacid sequence of SEQ ID NOs: 8 or 18. In some embodiments, the VLcomprises a CDR1 region comprising the amino acid sequence of SEQ IDNO:1; a CDR2 region comprising the amino acid sequence of SEQ ID NO:2;and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 3;and wherein the VH comprises a CDR1 region comprising the amino acidsequence of SEQ ID NO: 6; a CDR2 region comprising the amino acidsequence of SEQ ID NO: 7; and a CDR3 region comprising the amino acidsequence of SEQ ID NO:8. In some embodiments, the VL comprises a CDR1region comprising the amino acid sequence of SEQ ID NO: 11; a CDR2region comprising the amino acid sequence of SEQ ID NO: 12; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 13, and whereinthe VH comprises a CDR1 region comprising the amino acid sequence of SEQID NO: 16; a CDR2 region comprising the amino acid sequence of SEQ IDNO: 17; and a CDR3 region comprising the amino acid sequence of SEQ IDNO: 18. In some embodiments, the VL comprises a CDR1 region comprisingthe amino acid sequence of SEQ ID NO:21; a CDR2 region comprising theamino acid sequence of SEQ ID NO: 22; and a CDR3 region comprising theamino acid sequence of SEQ ID NO: 23, and wherein the VH comprises aCDR1 region comprising the amino acid sequence of SEQ ID NO: 26; a CDR2region comprising the amino acid sequence of SEQ ID NO: 27; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the VL comprises the amino acid sequence of SEQ IDNOs: 4, 14 or 24, and wherein the VH comprises the amino acid sequenceof SEQ ID NOs: 9, 19 or 29. In further embodiments, the VL comprises theamino acid sequence of SEQ ID NOs: 4 or 14, and wherein the VH comprisesthe amino acid sequence of SEQ ID NOs: 9 or 19.

In some embodiments:

-   (a) the VL comprises the amino acid sequence of SEQ ID NO: 4, and    the VH comprises SEQ ID NO: 9;-   (b) the VL comprises the amino acid sequence of SEQ ID NO: 14, and    the VH comprises SEQ ID NO: 19; or-   (c) the VL comprises the amino acid sequence of SEQ ID NO: 24, and    the VH comprises SEQ ID NO: 29.

In some embodiments, the antibody is humanized. In further embodiments,the antibody is labeled.

Also provided is a pharmaceutical composition comprising the monoclonalantibody of any one of the preceding embodiments and at least onepharmaceutical excipient.

Also provided is an isolated polynucleotide comprising at least one ofthe nucleic acid sequences of SEQ ID NOs: 5, 10, 15, 20, 25 or 30. Insome embodiments, the polynucleotide comprises:

(a) the nucleic acid sequences of SEQ ID NOs: 5 and 10;(b) the nucleic acid sequences of SEQ ID NOs: 15 and 20; or(c) the nucleic acid sequences of SEQ ID NOs: 25 and 30.

Provided is a method of treating a synucleopathic disease in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of the isolated monoclonal antibody ofany of the previous embodiments. In some embodiments, the synucleopathicdisease is at least one from the group consisting of Parkinson'sdisease, Parkinson's disease with dementia, Dementia with Lewy bodies,Alzheimer's disease, Down's syndrome, multiple-system atrophy, priondiseases, and other α-Syn related neurodegenerative disorders. Infurther embodiments, the antibody is provided to the subject as apharmaceutical composition. In yet further embodiments, the antibody isadministered parenterally to the subject.

Provided is a method of detecting a synucleopathic disease in a subject,the method comprising administering to the subject a labeled isolatedmonoclonal antibody of any of the preceding embodiments, and detectingpresence or absence of a complex of the labeled isolated monoclonalantibody with any α-Syn fibrils present in the subject, wherein, if thecomplex is detected, the subject has a synucleopathic disease.

Also provided is a method of detecting α-Syn fibrils in a sample, themethod comprising contacting the sample with a labeled isolatedmonoclonal antibody of any of the preceding embodiments, and detectingpresence or absence of a complex of the labeled isolated monoclonalantibody with any α-Syn fibrils present in the sample, wherein, if thecomplex is detected, α-Syn fibrils are present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thedrawings show specific embodiments. It should be understood, however,that the invention is not limited to the precise arrangements andinstrumentalities of the embodiments shown in the drawings.

FIG. 1A is a pair of graphs depicting the affinity of mAb Syn9063.26.22for human monomeric (top) and fibrillar (PFF) (bottom) α-Syn measured bysandwich ELISA. 384-well plates were coated with 0.3 μg of the indicatedmAb. After blocking, these were incubated with various concentrations ofα-Syn monomer or PFFs (expressed as monomer equivalent concentration) asindicated. MJF-R1 was used as a reporter antibody to determine affinityfor each form of α-Syn. FIG. 1B is a pair of graphs depicting theaffinity of mAb Syn9048.04.07 for human monomeric (top) and fibrillar(PFF) (bottom) α-Syn measured by sandwich ELISA. 384-well plates werecoated with 0.3 μg of the indicated mAb. After blocking, these wereincubated with various concentrations of α-Syn monomer or PFFs(expressed as monomer equivalent concentration) as indicated. MJF-R1 wasused as a reporter antibody to determine affinity for each form ofα-Syn. FIG. 1C is a pair of graphs depicting the affinity of mAbSyn9068.13.01 for human monomeric (top) and fibrillar (PFF) (bottom)α-Syn measured by sandwich ELISA. 384-well plates were coated with 0.3μg of the indicated mAb. After blocking, these were incubated withvarious concentrations of α-Syn monomer or PFFs (expressed as monomerequivalent concentration) as indicated. MJF-R1 was used as a reporterantibody to determine affinity for each form of α-Syn. Performance ofthree subclones show greater binding to human PFFs over monomer for9063.26.22 and 9048.04.07. For each of FIGS. 1A-1C, curve fitting wasdone using the software package GraphPad. All experiments were performedin triplicate and the numbers reported are the average ELISA values fromthose experiments.

FIG. 2 is a graph depicting data from an experiment in which primary WThippocampal neurons from embryonic CD1 mice were plated in 96-wellplates (17,500 cells per well). After 7 days in vitro (DIV), cells werepre-treated for 30 mins with either nonspecific mouse IgG (IgG), Syn211,or Syn9063.26.22 at the indicated mAb:PFF ratios. Cells were thentreated with PBS, human wild-type hWT α-Syn PFFs, or mouse wild-type(mWT) α-Syn PFFs (0.125 μg/well) and fixed 7 days later in 4% PFA.Neurons were immunostained with mAb 81A to detect p-α-Syn positivepathology. The graph shows mean levels of p-α-Syn pathology fromtriplicate samples from 2 independent experiments. Horizontal linesrepresent the extent of p-α-Syn positive pathology in neurons treatedonly with mWT α-Syn PFFs (M) or hWT α-Syn PFFs (H).

FIG. 3 is a graph depicting data from an experiment in which primaryhippocampal neurons from embryonic CD1 mice were plated in 96-wellplates (17,500 cells per well). After 7 days in vitro (DIV), cells werepre-treated for 30 mins with either nonspecific mouse IgG (IgG), Syn211,or Syn9048.04.07 at the indicated mAb:PFF ratios. Cells were thentreated with PBS, hWT α-Syn pffs or mWT α-Syn pffs (0.125 μg/well each)and fixed 7 days later in 4% PFA. Neurons were immunostained with mAb81A to detect p-α-Syn positive pathology. The graph shows mean levels ofp-α-Syn pathology after different mAb treatments. Horizontal linesrepresent the extent of p-α-Syn positive pathology in neurons treatedonly with mWT α-Syn PFFs (M) or hWT α-Syn PFFs (H).

FIGS. 4A and 4B are a pair of graphs depicting data from an experimentin which primary hippocampal neurons from embryonic CD1 mice were platedin 96-well plates (17,500 cells per well). After 7 days in vitro (DIV),cells were pre-treated for 30 mins with either nonspecific mouse IgG(IgG) or Syn9068.13.01 at the indicated mAb:PFF ratios. Cells were thentreated with PBS, hWT α-Syn PFFs (FIG. 4A), or mWT, or hWT α-Syn PFFs(FIG. 4B; 0.125 μg/well each) and fixed 7 days later in 4% PFA. Neuronswere immunostained with mAb 81A to detect p-α-Syn positive pathology.The graphs show mean levels of p-α-Syn pathology after different mAbtreatments.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, analytical chemistry, immunology, andnucleic acid chemistry and hybridization are those well-known andcommonly employed in the art. Standard techniques or modificationsthereof are used for chemical syntheses and chemical analyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “α-Synuclein” or “α-Syn” or “α-syn” refers to aprotein that is expressed mainly in brain tissues and is primarilylocated at the presynaptic terminal of neurons. In certain embodiments,the invention contemplates human α-Syn, which has the sequence SEQ IDNO: 31:

        10         20         30         40MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV        50         60         70         80GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK        90        100        110        120TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP        130        140DNEAYEMPSE EGYQDYEPEA

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a concentration, a temporal duration, and the like,the term “about” is meant to encompass variations of ±20% or ±10%, morepreferably ±5%, even more preferably ±1%, and still more preferably±0.1% from the specified value, as such variations are appropriate toperform the disclosed methods.

As used herein, the term “affinity” for a molecule towards anotherrefers to the degree (or tightness) of binding between the twomolecules. A higher affinity means tighter binding between the twomolecules. Affinity can be quantified in terms of dissociation constant(or K_(d)), where a K_(d) value that is lower in magnitude (closer tozero) indicates a higher affinity.

An “amino acid” as used herein is meant to include both natural andsynthetic amino acids, and both D and L amino acids. “Standard aminoacid” means any of the twenty L-amino acids commonly found in naturallyoccurring peptides. “Nonstandard amino acid residues” means any aminoacid, other than the standard amino acids, regardless of whether it isprepared synthetically or derived from a natural source. As used herein,“synthetic amino acid” also encompasses chemically modified amino acids,including but not limited to salts, amino acid derivatives (such asamides), and substitutions. Amino acids contained within the peptides,and particularly at the carboxy- or amino-terminus, can be modified bymethylation, amidation, acetylation or substitution with other chemicalgroups which can change a peptide's circulating half life withoutadversely affecting activity of the peptide. Additionally, a disulfidelinkage may be present or absent in the peptides.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule able to specifically bind to a specific epitope on an antigen.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions of intactimmunoglobulins. Antibodies are typically tetramers of immunoglobulinmolecules. The antibodies in the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Faband F(ab)₂, as well as single chain antibodies (scFv), camelidantibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426). As used herein, a“neutralizing antibody” is an immunoglobulin molecule that binds to andblocks the biological activity of the antigen.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated or synthesized, or can bederived from a biological sample. Such a biological sample can include,but is not limited to a tissue sample, a tumor sample, a cell or abiological fluid.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene that are homologous with or complementary to, respectively,the coding region of an mRNA molecule produced by transcription of thegene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule that are matched with an anti-codon regionof a transfer RNA molecule during translation of the mRNA molecule orthat encode a stop codon. The coding region may thus include nucleotideresidues corresponding to amino acid residues not present in the matureprotein encoded by the mRNA molecule (e.g., amino acid residues in aprotein export signal sequence).

“Complementary” as used herein to refer to a nucleic acid, refers to thebroad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. Itis known that an adenine residue of a first nucleic acid region iscapable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an antiparallel fashion, at least about 50%,and preferably at least about 75%, at least about 90%, or at least about95% of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. More preferably,all nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

The term “delivery vehicle” is used herein as a generic reference to anydelivery vehicle capable of delivering a compound to a subject,including, but not limited to, dermal delivery vehicles and transdermaldelivery vehicles.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein, effective toachieve a particular biological result. Such results may include, butare not limited to, treatment of a disease or condition as determined byany means suitable in the art.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “fragment,” as applied to a protein or peptide,refers to a subsequence of a larger protein or peptide. A “fragment” ofa protein or peptide can be at least about 20 amino acids in length; forexample at least about 50 amino acids in length; at least about 100amino acids in length, at least about 200 amino acids in length, atleast about 300 amino acids in length, and at least about 400 aminoacids in length (and any integer value in between). As used herein, anantibody fragment refers to active fragments thereof, i.e., fragmentshaving the same characteristics that are used for the definition of anantibody according to the invention, in certain embodiments highaffinity for α-Syn fibrils (composed of misfolded α-Syn) and low or highbinding affinity to α-Syn monomers. For convenience when the termantibody is used, fragments thereof exhibiting the same characteristicare also being considered.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides, at least about 1000 nucleotides to about 1500nucleotides; or about 1500 nucleotides to about 2500 nucleotides; orabout 2500 nucleotides (and any integer value in between).

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

An “individual”, “patient” or “subject”, as that term is used herein,includes a member of any animal species including, but are not limitedto, birds, humans and other primates, and other mammals includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and dogs. Preferably, the subject is a human.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

By “nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone linkages, and combinations of suchlinkages. The term nucleic acid also specifically includes nucleic acidscomposed of bases other than the five biologically occurring bases(adenine, guanine, thymine, cytosine and uracil).

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 60 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound of the invention with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

“Pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “protein”, “peptide” and “polypeptide” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. The term “peptide bond”means a covalent amide linkage formed by loss of a molecule of waterbetween the carboxyl group of one amino acid and the amino group of asecond amino acid. A protein or peptide must contain at least two aminoacids, and no limitation is placed on the maximum number of amino acidsthat may comprise the sequence of a protein or peptide. Polypeptidesinclude any peptide or protein comprising two or more amino acids joinedto each other by peptide bonds. As used herein, the term refers to bothshort chains, which also commonly are referred to in the art aspeptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. “Proteins” include, for example, biologicallyactive fragments, substantially homologous proteins, oligopeptides,homodimers, heterodimers, variants of proteins, modified proteins,derivatives, analogs, and fusion proteins, among others. The proteinsinclude natural proteins, recombinant proteins, synthetic proteins, or acombination thereof. A protein may be a receptor or a non-receptor.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “therapeutic” as used herein means a treatment and/orprophylaxis.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a subject or administering an agent orcompound to reduce the frequency and/or severity with which symptoms areexperienced. As used herein, “alleviate” is used interchangeably withthe term “treat.”

As used herein, “treating a disease, disorder or condition” meansreducing the frequency or severity with which a symptom of the disease,disorder or condition is experienced by a subject. Treating a disease,disorder or condition may or may not include complete eradication orelimination of the symptom.

The following abbreviations are used herein: CDR,complementary-determining region; DLB, dementia with Lewy bodies; MSA,multiple system atrophy; PD, Parkinson's disease; VH, heavy chainvariable region; VL, light chain variable region.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

This disclosure is generally directed to certain monoclonal (mouse)antibodies, or fragments thereof, that recognize α-Syn, a protein thatis misfolded in Parkinson's disease and related neurodegenerativedisorders known as synucleinopathies. Whereas the majority of antibodiesagainst α-Syn have been generated using the soluble monomeric (i.e.native) form of the protein as an antigen, the present antibodies wereraised against recombinant α-Syn that was misfolded into amyloid fibrilsin vitro and resembles the pathological form found in human disease. Itis demonstrated herein that the antibodies of the invention recognizeα-Syn that accumulates in the brains of humans with Parkinson's disease.In certain embodiments, the antibodies of the invention recognize aconformational epitope comprising amino acids 110-120 of α-Syn. In otherembodiments, the antibodies of the invention recognize a conformationalepitope comprising amino acids 120-130 of α-Syn.

In certain embodiments, the antibodies of the invention showpreferential binding towards the pathological form of α-Syn (i.e.,fibrils) compared to the native (i.e., monomeric) form. In otherembodiments, the antibodies of the invention reduce formation ofpathological α-Syn inclusions/fibrils that normally form in culturedneurons that are exposed to recombinant α-Syn fibrils. In yet otherembodiments, the antibodies of the invention detect pathological α-Synfibrils. In yet other embodiments, the antibodies of the invention areused as therapeutics for decreasing the development/spread ofpathological α-Syn fibrils in synucleinopathies. In yet otherembodiments, the antibodies of the invention do not cross-react with Tauand/or beta-amyloid protein.

In certain embodiments, the antibodies of the invention bind to α-Synfibrils with a dissociation constant K_(d) equal to or less than about10⁻⁶ M, about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, orabout 10⁻¹¹ M. In other embodiments, the antibodies of the inventionbind to α-Syn monomers with an affinity that at least about 10 times, 30times, 100 times, 300 times, or 1000 times lower than the affinity ofthe antibodies for α-Syn fibrils. In yet other embodiments, theantibodies of the invention bind to α-Syn monomers with a dissociationconstant K_(d) equal to or higher than about 10⁻¹⁰ M, about 10⁻⁹ M,about 10⁻⁸ M, about 10⁻⁷ M, about 10⁻⁶ M, about 10⁻⁵ M, about 10⁻⁴ M, orabout 10⁻³ M. In yet other embodiments, the antibodies of the inventionbind with nearly equal affinity to α-Syn fibrils and monomers. In yetother embodiments, the antibodies of the invention bind with nearlyequal affinity to α-Syn fibrils and monomers, with a dissociationconstant K_(d) equal to or less than about 10⁻⁷ M, about 10⁻⁸ M, about10⁻⁹ M, about 10⁻¹⁰ M, or about 10⁻¹¹ M. Binding affinities of theantibodies can be determined by using a variety of methods recognized inthe art, including methods described elsewhere herein, such as but notlimited to isothermal calorimetry, surface plasmon resonance,immunoassays such as ELISA or RIAs, and the like.

Compositions Comprising Antibodies

In one aspect, the invention comprises isolated monoclonal antibodiesthat selectively bind α-Syn in the fibrillar conformation, and/or bindboth soluble and fibrillar α-Syn with high affinity. In certainembodiments, the antibody comprises a heavy chain. In other embodiments,the heavy chain comprises three complementary-determining regions (CDR),namely CDR1, CDR2 and CDR3. In yet other embodiments, the light chaincomprises three complementary-determining region (CDR), namely CDR1,CDR2 and CDR3.

In certain embodiments, the monoclonal antibody is derived fromhybridoma Syn9063.26.22 with light and heavy variable chains having thesequences shown below:

VL (Variable Light) MHC978LC.2\;M13 CDR Analysis SSVSY . . . _ DTS_QQWRSYPPT SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3Amino Acid Sequence in FASTA format > MHC978LC.2\;M13 SEQ ID NO: 4QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSKLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWRSYPPTFGAG TKLELKNucleotide Sequence in FASTA format > MHC978LC.2\;M13 SEQ ID NO: 5CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAAGCTGGCTTCTGGCGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGAAGTTACCCACCCACGTTCGGTGCTGGG ACCAAGCTGGAGCTGAAAVH (Variable Heavy) MHC978HC.1\;M13 CDR Analysis GFSLTSYG . . ._ IWSGGST . . . _ARTFTTSTSAWFAY SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8Amino Acid Sequence in FASTA format >MHC978.HC.1\;M13 SEQ ID NO: 9QVQLKQSGPGLVQPSQSQSLTCTVSGFSLTSYGVHWVRQPLGKGLEWLGVIWSGGSTDYNAAFISRLSIRKDNSKSQVFFKMNSLQADDTAIYYCARTFT TSTSAWFAYWGQGTLVTVSANucleotide Sequence in FASTA format > MHC978HC.1\;M13 SEQ ID NO: 10CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCAGTCCCTGACCTGCACAGTCTCTGGTTTCTCATTAACTAGTTATGGCGTACACTGGGTTCGCCAGCCTCTAGGAAAGGGTCTGGAGTGGCTGGGAGTGATCTGGAGTGGTGGAAGCACAGACTATAATGCTGCTTTCATATCCAGACTGAGCATCAGGAAGGACAACTCCAAGAGCCAAGTCTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGAACCTTTACTACGTCTACCTCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCAC TGTCTCTGCA

In certain embodiments, the monoclonal antibody is derived fromhybridoma Syn9048.04.07 with the light and heavy variable chains havingthe sequences shown below:

VL (Variable Light) MHC992LC.1\;M13 CDR Analysis QSVLYSSNQKNY _WAS_HQYLSLFT SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13Amino Acid Sequence in FASTA format >MHC992LC.1\;M13 SEQ ID NO: 14NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKMLIYWASFRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSL FTFGSGTKLEIKNucleotide Sequence in FASTA format >MHC992LC.1\;M13 SEQ ID NO: 15AACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAAATGCTGATCTACTGGGCATCCTTTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATACCTCTCCTTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA VH (Variable Heavy) MHC992HC.2\;M13CDR Analysis GYTFTTFY . . . IYPVNVKI . . . VRGGRGLDY SEQ ID NO: 16SEQ ID NO: 17 SEQ ID NO: 18Amino Acid Sequence in FASTA format >MHC992HC.2\;M13 SEQ ID NO: 19QVQLQQSGPELVKPGASVRISCKASGYTFTTFYLHWVKQRPGQGLEWIGWIYPVNVKIKYSERFKGKATLTADKSSSTAYMQLGSLTSEDSAVYFCVRGG RGLDYWGQGTTLTVSSNucleotide Sequence in FASTA format >MHC992HC.2\;M13 SEQ ID NO: 20CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGCGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAACCTTCTATTTACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGTAAATGTTAAAATTAAGTACAGTGAGAGGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCGGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGTAAGAGGGGGGAGGGGACTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

In certain embodiments, the monoclonal antibody is derived fromhybridoma Syn9068.13.01 with the light and heavy variable chains havingthe sequences shown below:

VL (Variable Light) MHC993LC.3\;M13 CDR Analysis SSVTY . . . DTSQQWSSNPPT SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23Amino Acid Sequence in FASTA format >MHC993LC.3\;M13 SEQ ID NO: 24QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGG TKLEIRNucleotide Sequence in FASTA format >MHC993LC.3\;M13 SEQ ID NO: 25CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAACTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCACGTTCGGAGGGGGG ACCAAGCTGGAAATAAGAVH (Variable Heavy) MHC993HC.1\;M13 CDR Analysis GFTFNTYA . . .IRNKSNNYAT VRGGLSPFDY SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28Amino Acid Sequence in FASTA format >MHC993HC.1\;M13 SEQ ID NO: 29EVHLVESGGGLVQPKGSLKLSCAASGFTFNTYAMHWVRQAPGKGLEWVARIRNKSNNYATYYADSVKDRFTISRDDSQSMLFLQMDNLKTEDTAIYYCVR GGLSPFDYWGQGTTLTVSSNucleotide Sequence in FASTA format >MHC993HC.1\;M13 SEQ ID NO: 30GAGGTACACCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCTACGCCATGCACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAATAAAAGTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTTTCTGCAAATGGACAACTTGAAAACTGAGGACACAGCCATATATTACTGTGTGAGAGGGGGGTTATCTCCCTTTGACTACTGGGGCCAAGGCACCACACTCACAGT CTCCTCA

In certain embodiments, the antibody comprises an immunoglobulin lightchain variable region (VL) comprising the amino acid sequence of SEQ IDNOs: 4, 14 or 24. In other embodiments, the antibody comprises a VLcomprising the amino acid sequence of SEQ ID NOs: 4 or 14.

In certain embodiments, the antibody comprises an immunoglobulin heavychain variable region (VH) comprising the amino acid sequence of SEQ IDNOs: 9, 19 or 29. In other embodiments, the antibody comprises a VHcomprising the amino acid sequence of SEQ ID NOs: 9 or 19.

In certain embodiments, the antibody comprises a VL comprising the aminoacid sequence of SEQ ID NO:4, and a VH comprising SEQ ID NO: 9. In otherembodiments, the antibody comprises a VL comprising the amino acidsequence of SEQ ID NO: 14, and a VH comprising SEQ ID NO: 19. In yetother embodiments, the antibody comprises a VL comprising the amino acidsequence of SEQ ID NO: 24, and a VH comprising SEQ ID NO: 29.

In certain embodiments, the antibody comprises a VL comprising: a CDR1region comprising the amino acid sequence of SEQ ID NOs: 1, 11 or 21; aCDR2 region comprising the amino acid sequence of SEQ ID NOs: 2, 12 or22; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs:3, 13 or 23. In other embodiments, the antibody comprises a VLcomprising: a CDR1 region comprising the amino acid sequence of SEQ IDNOs: 1 or 11; a CDR2 region comprising the amino acid sequence of SEQ IDNOs: 2 or 12; and a CDR3 region comprising the amino acid sequence ofSEQ ID NOs: 3 or 13.

In certain embodiments, the antibody comprises a VL comprising: a CDR1region comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 3. In otherembodiments, the antibody comprises a VL comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 11; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 12; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 13. In yet otherembodiments, the antibody comprises a VL comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 21; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 23.

In certain embodiments, the antibody comprises a VH comprising: a CDR1region comprising the amino acid sequence of SEQ ID NOs: 6, 16 or 26; aCDR2 region comprising the amino acid sequence of SEQ ID NOs: 7, 17 or27; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs:8, 18 or 28. In other embodiments, the antibody comprises a VHcomprising: a CDR1 region comprising the amino acid sequence of SEQ IDNOs: 6 or 16; a CDR2 region comprising the amino acid sequence of SEQ IDNOs: 7 or 17; and a CDR3 region comprising the amino acid sequence ofSEQ ID NOs: 8 or 18.

In certain embodiments, the antibody comprises a VH comprising: a CDR1region comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 8. In otherembodiments, the antibody comprises a VH comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 16; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 18. In yet otherembodiments, the antibody comprises a VH comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 26; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 27; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 28.

In certain embodiments, the antibody comprises a VL comprising: a CDR1region comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 3; and a VH comprising:a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6; a CDR2region comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 8. In otherembodiments, the antibody comprises a VL comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 11; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 12; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 13; a VH comprising: aCDR1 region comprising the amino acid sequence of SEQ ID NO: 16; a CDR2region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 18. In yet otherembodiments, the antibody comprises a VL comprising: a CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 21; a CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 23; and a VHcomprising: a CDR1 region comprising the amino acid sequence of SEQ IDNO: 26; a CDR2 region comprising the amino acid sequence of SEQ ID NO:27; and a CDR3 region comprising the amino acid sequence of SEQ ID NO:28.

The invention further provides isolated polynucleotides (including RNAand/or DNA) encoding the antibodies or antigen binding fragmentsthereof, for example a nucleic acid encoding for one or more CDRs, or avariable heavy chain or variable light chain region of the α-Synantibodies of the invention. Nucleic acid includes DNA and RNA.

In certain embodiments, the antibody has a VL encoded by the nucleicacid sequence of SEQ ID NOs: 5, 15 or 25. In other embodiments, theantibody has a VL encoded by the nucleic acid sequence of SEQ ID NOs: 5or 15. In yet other embodiments, the antibody has a VH encoded by thenucleic acid sequence of SEQ ID NOs: 10, 20 or 30. In yet otherembodiments, the antibody has a VH encoded by the nucleic acid sequenceof SEQ ID NOs: 10 or 20.

In certain embodiments, the invention provides an isolatedpolynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 5, 15or 25. In other embodiments, the invention provides an isolatedpolynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 5 or15. In yet other embodiments, the invention provides an isolatedpolynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 10,20 or 30. In yet other embodiments, the invention provides an isolatedpolynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 10 or20.

In certain embodiments, the invention provides an isolatedpolynucleotide comprising a nucleic acid encoding the amino acidsequence of SEQ ID NOs: 4, 14 or 24. In other embodiments, the inventionprovides an isolated polynucleotide comprising a nucleic acid encodingthe amino acid sequence of SEQ ID NOs: 4 or 14. In yet otherembodiments, the invention provides an isolated polynucleotidecomprising a nucleic acid encoding the amino acid sequence of SEQ IDNOs: 9, 19 or 29. In yet other embodiments, the invention provides anisolated polynucleotide comprising a nucleic acid encoding the aminoacid sequence of SEQ ID NOs: 9 or 19.

The invention further provides a host cell comprising the expressionvector. In certain embodiments, the host cell is isolated. In otherembodiments, the host cell is a non-human cell. The expression vectorcan comprise nucleic acid sequences that direct and/or controlexpression of the inserted polynucleotide. Such nucleic acid sequencescan include regulatory sequence, including promoter sequences,terminator sequences, polyadenylation sequences, and enhancer sequences.Systems for cloning and expression of a polypeptide in a variety ofcells are well known in the art.

The antibody of the invention can be a mammalian antibody, such asprimate, human, rodent, rabbit, ovine, porcine or equine antibody. Theantibody can be any class or isotype antibody, for example IgM or IgG.In certain embodiments, the antibody is IgG.

The invention further provides a kit comprising an antibody of theinvention. The antibody may be an intact immunoglobulin molecule orfragment thereof such as Fab, F(ab)2 or Fv fragment. The antibody can belabelled as described elsewhere herein. The kit can be for use in amethod of determining whether a subject has a neurodegenerative disease,and/or for treating a subject afflicted or thought to be afflicted witha neurodegenerative disease. The kit can further any other reagent orinstrument that is required to implement a method of the invention, suchas a buffer, an applicator, and the like.

The non-limiting generation of these antibodies is illustrated inExample 1. Data showing preferential binding of α-Syn fibrils overmonomer is presented in FIGS. 1A-1B, high affinity binding to both α-Synfibrils and monomer in FIG. 1C, and these properties are furtherdescribed in FIGS. 2-4. In certain embodiments, the invention comprisespharmaceutical compositions comprising each of these antibodies incombination with one or more pharmaceutically acceptable excipients. Insome embodiments the pharmaceutical composition is formulated forparenteral delivery. In other embodiments, the antibodies are humanized.

Method of Treating a Synucleopathic Disease

In another aspect, the invention provides a method of treating asynucleopathic disease comprising administering a therapeuticallyeffective amount of an isolated monoclonal antibody of the invention toa patient. In certain embodiments, the antibody is humanized. In otherembodiments, the antibody is administered as a pharmaceuticalcomposition.

The monoclonal antibodies described above may be used to treatsynucleopathic disease by reducing α-Syn pathology in neurons induced byα-Syn fibrils, as shown in FIGS. 3 and 4 and further described inExample 3. In certain embodiments, the neurodegenerative disordersassociated with α-Syn include but are not limited to Parkinson'sdisease, dementia (such as Parkinson's disease with dementia and/ordementia with Lewy bodies), Alzheimer's disease, Down's syndrome,multiple-system atrophy, prion diseases, and other α-Syn relatedneurodegenerative disorders. The antibody can be administeredsystemically or directly to the site where α-Syn fibrils, e.g. a Lewybody, are observed or thought to be present. In a non-limiting example,the antibody can be administered by injection into a blood vesselsupplying the brain or into the brain itself. The subject can be amammal, such as a human or a non-human mammal.

In some embodiments, administration of the antibody may result in apreservation in dopamine production in the brain. In some embodiments,the preservation in dopamine production is an increase in striataldopamine production.

Methods of Detecting a Synucleopathic Disease

In yet another aspect, the invention provides methods of detectingsynucleopathic disease in a patient. In other embodiments, theantibodies of the invention can be used as diagnostic tools forneurodegenerative disorders associated with α-Syn, including but notlimited to Parkinson's disease, dementia (such as Parkinson's diseasewith dementia and/or dementia with Lewy bodies), Alzheimer's disease,Down's syndrome, multiple-system atrophy, prion diseases, and otherα-Syn related neurodegenerative disorders.

In certain embodiments, the method of detecting synucleopathic diseasein a subject comprises the steps of administering a labeled, isolatedmonoclonal antibody of the invention to the subject, and detecting thepresence of absence of a complex between any α-Syn fibrils in thesubject and the antibody. If the complex is present, that indicates thatα-Syn fibrils exist in the subject. In certain embodiments, if α-Synfibrils are present in the subject, the subject has a neurodegenerativedisease. In other embodiments, if α-Syn fibrils are not present in thesubject, the subject does not have a neurodegenerative disease. In yetother embodiments, if the subject has a neurodegenerative disease, theindividual is counseled to undergo therapy and/or pharmacologicaltreatment for the neurodegenerative disease. In yet other embodiments,if the subject has a neurodegenerative disease, the individual isprovided therapy and/or pharmacological treatment for theneurodegenerative disease.

In certain embodiments, the method further comprises comparing the levelof antibody/α-Syn fibrils complex formed in the subject with the levelof antibody/α-Syn fibrils complex formed in a reference subject. Thereference subject can be a subject known not to have α-Syn fibrils, asubject known to have detectable α-Syn fibrils, and/or a subject knownto have a certain level of α-Syn fibrils. The reference subject canfurther be the same subject being treated or evaluated, butcorresponding to an earlier α-Syn fibrils detection experiment, as a wayto evaluate disease progression and/or treatment efficacy in thesubject.

In yet another aspect, the invention provides methods of detecting α-Synfibrils in a sample. In certain embodiments, the antibodies of theinvention can be used as diagnostic tools for detecting the presence ofα-Syn fibrils in a sample.

In certain embodiments, the method of detecting α-Syn fibrils in asample (for example, from a subject) comprises the steps of contactingthe sample with a labeled, isolated monoclonal antibody of theinvention, and detecting the presence or absence of a complex betweenany α-Syn fibrils in the sample and the antibody. If the complex isdetected, that indicates the presence of α-Syn fibrils in the sample.The sample can be, in non-limiting examples, cerebrospinal fluid (CSF),blood, urine, saliva, or tissues from brain, gut, colon, skin, orsalivary gland. In certain embodiments, the sample is a CSF sampleand/or a brain tissue sample. In other embodiments, the sample is usedas is after being removed from the subject. In other embodiments, thesample is pre-treated being used within the present methods.

In certain embodiments, the method further comprises comparing the levelof antibody-α-Syn fibrils complex formed in the sample with the level ofantibody-α-Syn fibrils complex formed in a reference sample. Thereference sample can be from a subject known not to have α-Syn fibrils,a subject known to have detectable α-Syn fibrils, and/or a subject knownto have a certain level of α-Syn fibrils. The reference sample canfurther be from the same subject being treated or evaluated, butcorresponding to an earlier α-Syn fibrils detection, as a way toevaluate disease progression and/or treatment efficacy in the subject.

In certain embodiments, the level of α-Syn fibrils detected in a subjector in a sample from a subject correlates with severity or progression ofa neurodegenerative disease in the subject. In other embodiments, themethods of the invention can be used to monitor severity or progressionof a neurodegenerative disease in the subject. In yet other embodiments,the methods of the invention can be used to monitor effectiveness of atherapy and/or pharmacological intervention in a subject afflicted orbelieved to be afflicted with a neurodegenerative disease.

Methods for detecting formation of a complex between the antibody andα-Syn fibrils comprise, but are not limited to, radioimmunoassay,enzyme-linked immunosorbant assay (ELISA), sandwich immunoassay,fluorescent immunoassay, precipitation reaction, gel immunodiffusionassay, agglutination assay, protein A immunoassay, immunoelectrophoresisassay, electrophoresis, western blotting, or any other technique knownin the art.

The antibodies of the invention can be combined with a label and used todetect α-Syn fibrils in a patient or in a sample. Methods of labelingantibodies are known in the art and a variety of approaches may beemployed. In certain embodiments the label is a radiolabel, such as butnot limited to F¹⁸, I¹²³, In¹¹¹, I¹³¹, C¹⁴, H³, Tc^(99m), P³², I¹²⁵,Ga⁶⁸ and the like. In other embodiments, the label is a fluorescentlabel, such as but not limited to fluorescein, rhodamine and the like.In yet other embodiments, the label is a contrast agent, such as but notlimited to gadolinium (Gd), dysprosium and iron, magnetic agents, andthe like. Other labels include nuclear magnetic resonance active labels,positron emitting isotopes detectable by a PET scanner, chemiluminescentand enzymatic markers. Non-limiting imaging techniques include electronmicroscopy, confocal microscopy, light microscopy, positron emissiontomography (PET), gamma-scintigraphy, magnetic resonance imaging (MRI),functional magnetic resonance imaging (FMRI), magnetoencephalography(MEG), and single photon emission computerized tomography (SPECT). Inyet other embodiments, the label is on a secondary antibody that binds aprimary antibody comprising the above described sequences.

Administration/Dosage/Formulations

Administration of the compounds and/or compositions of the presentinvention to a patient, preferably a mammal, more preferably a human,may be carried out using known procedures, at dosages and for periods oftime effective to perform an imaging method contemplated in theinvention. An effective amount of the therapeutic compound necessary foradequate signal for imaging may vary according to factors such as thestate of a disease or disorder in the patient; the age, sex, and weightof the patient; and the equipment used to detect the compound of theinvention. One of ordinary skill in the art would be able to study therelevant factors and make the determination regarding the effectiveamount of the therapeutic and/or imaging compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve successful imagingfor a particular patient, composition, and mode of administration,without being toxic to the patient.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise an effective amount of a compound of the invention and apharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In certain embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In certain embodiments, a dose of acompound of the invention used in compositions described herein is lessthan about 10,000 mg, or less than about 8,000 mg, or less than about6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, orless than about 2,000 mg, or less than about 1,000 mg, or less thanabout 500 mg, or less than about 200 mg, or less than about 50 mg.Similarly, in certain embodiments, a dose of a second compound asdescribed herein is less than about 1,000 mg, or less than about 800 mg,or less than about 600 mg, or less than about 500 mg, or less than about400 mg, or less than about 300 mg, or less than about 200 mg, or lessthan about 100 mg, or less than about 50 mg, or less than about 40 mg,or less than about 30 mg, or less than about 25 mg, or less than about20 mg, or less than about 15 mg, or less than about 10 mg, or less thanabout 5 mg, or less than about 2 mg, or less than about 1 mg, or lessthan about 0.5 mg, and any and all whole or partial increments thereof.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans) buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multidose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In certain embodiments ofa formulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Application Nos.2003/0147952; 2003/0104062; 2003/0104053; 2003/0044466; 2003/0039688;and 2002/0051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, practice the claimed methods of the presentinvention. The following working examples therefore, specifically pointout the preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure.

Example 1: Generation of Monoclonal Antibodies Against Aggregated α-Syn

Syn9063.26.22, Syn9048.04.07 and Syn9068.13.01 are mouse monoclonalantibodies (mAbs) generated from mice immunized with aggregatedrecombinant WT human α-Syn bearing strain-B conformation. Preparation ofthis antigen is described in Guo et al., 2013, Cell. 2013 154(1):103-17.Hybridomas were generated, and subcloned twice by single cell dilution.Clonality was confirmed by sequencing. RNA was extracted from subclonesand amplified using RACE/PCR. Variable regions (H+L) were sequencedusing VBASE2 to assign complementarity determining regions (CDRs).

The properties of 9000 series mAbs are presented below in Table 1:

TABLE 1 Syn9063. Syn9048. Syn9068. Property/criteria 26.22 04.07 13.01Epitope 110-120 110-120 120-130 Isotype IgG2b IgG1 IgG1 Recognition ofHu vs. Ms 1.1-fold 1.4-fold 1.0-fold αSyn (direct ELISA) Selectivebinding to PFFs 100-fold 30-fold none (sandwich ELISA) Inhibition inneuron assay ↓80% ↓>95% ↓85% (Hu PFFs); see FIGS. 2-4 Inhibition inneuron assay ↓50% ↓>95% ~90% (Ms PFFs); see FIGS. 2-4 IHC on humantissue Strong Strong Strong IHC on mouse tissue Strong Strong Strong HuPFFs, recombinant human preformed αSyn fibrils; Ms PFF, recombinantmouse preformed αSyn fibrils; IHC, immunohistochemistry

Example 2: Affinity of Syn9063.26.22, Syn9048.04.07, 9068.13.01 forα-Syn Monomer vs. Fibrils

The affinity of three mAbs from the 9000 series for human monomeric andfibrillar (PFF) α-Syn was measured by sandwich ELISA. 384-well plateswere coated with 0.3 ug of the indicated mAb. After blocking, these wereincubated with various concentrations of α-Syn monomer or PFFs asindicated. MJR-R1 was used as a reporter antibody to determine affinityfor each form of α-Syn. Subclones 9063.26.22 and 9048.04.07 show greaterbinding to human PFFs than monomer. Curve fitting was done using thesoftware package GraphPad. All experiments were performed in triplicateand the numbers reported are the average ELISA values from thoseexperiments. The data are presented in FIGS. 1A-1C.

Example 3: Syn9063.26.22, Syn9048.04.07 and Syn 9068.13.01 Reduce α-SynPathology in Neurons Induced by α-Syn Fibrils

Primary WT hippocampal neurons from CD1 mice were plated in 96-wellplates (17,500 cells per well). After 7 days in vitro (DIV), cells werepre-treated for 30 mins with either nonspecific mouse IgG (IgG), Syn211,Syn9063.26.22, Syn9048.04.07 at the indicated mAb:α-Syn PFF ratios.Cells were then treated with PBS, hWT α-Syn PFFs, or mWT α-Syn PFFs(0.125 μg/well) and fixed 7 days later in 4% PFA. Neurons wereimmunostained with mAb 81A to detect p-α-Syn positive pathology. Graphshows mean levels of p-α-Syn pathology from triplicate samples from 2independent experiments. The data are presented in FIG. 2(Syn9063.26.22) or FIG. 3 (Syn9048.04.07).

Primary WT neurons from CD1 mice were plated in 96-well plates (17,500cells per well). After 7 days in vitro (DIV), cells were pre-treated for30 mins with either nonspecific mouse IgG (IgG) or Syn9068.13.01 at theindicated mAb:α-Syn PFF ratios. Cells were then treated with PBS, hWTα-Syn PFFs (upper panel), or mWT α-Syn PFFs (lower panel; 0.125 μg/welleach) and fixed 7 days later in 4% PFA. Neurons were immunostained withmAb 81A to detect p-α-Syn positive pathology. Graph shows mean levels ofp-α-Syn pathology after different mAb treatments. The data are presentedin FIGS. 4A-4B.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. An isolated monoclonal antibody comprising alight chain variable region (VL) and a heavy chain variable region (VH),wherein the VL comprises a CDR1 region comprising the amino acidsequence of SEQ ID NOs: 1, 11 or 21; a CDR2 region comprising the aminoacid sequence of SEQ ID NOs: 2, 12 or 22; and a CDR3 region comprisingthe amino acid sequence of SEQ ID NOs: 3, 13 or 23, and wherein the VHcomprises a CDR1 region comprising the amino acid sequence of SEQ IDNOs: 6, 16 or 26; a CDR2 region comprising the amino acid sequence ofSEQ ID NOs: 7, 17 or 27; and a CDR3 region comprising the amino acidsequence of SEQ ID NOs: 8, 18 or
 28. 2. The monoclonal antibody of claim1, wherein the VL comprises a CDR1 region comprising the amino acidsequence of SEQ ID NOs: 1 or 11; a CDR2 region comprising the amino acidsequence of SEQ ID NOs: 2 or 12; and a CDR3 region comprising the aminoacid sequence of SEQ ID NOs: 3 or 13, and wherein the VH comprises aCDR1 region comprising the amino acid sequence of SEQ ID NOs: 6 or 16; aCDR2 region comprising the amino acid sequence of SEQ ID NOs: 7 or 17;and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 8 or18.
 3. The monoclonal antibody of claim 1, wherein the VL comprises aCDR1 region comprising the amino acid sequence of SEQ ID NO: 1; a CDR2region comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 3; and whereinthe VH comprises a CDR1 region comprising the amino acid sequence of SEQID NO: 6; a CDR2 region comprising the amino acid sequence of SEQ ID NO:7; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 8.4. The monoclonal antibody of claim 1, wherein the VL comprises a CDR1region comprising the amino acid sequence of SEQ ID NO: 11; a CDR2region comprising the amino acid sequence of SEQ ID NO: 12; and a CDR3region comprising the amino acid sequence of SEQ ID NO: 13, and whereinthe VH comprises a CDR1 region comprising the amino acid sequence of SEQID NO: 16; a CDR2 region comprising the amino acid sequence of SEQ IDNO: 17; and a CDR3 region comprising the amino acid sequence of SEQ IDNO:
 18. 5. The monoclonal antibody of claim 1, wherein the VL comprisesa CDR1 region comprising the amino acid sequence of SEQ ID NO: 21; aCDR2 region comprising the amino acid sequence of SEQ ID NO: 22; and aCDR3 region comprising the amino acid sequence of SEQ ID NO: 23, andwherein the VH comprises a CDR1 region comprising the amino acidsequence of SEQ ID NO: 26; a CDR2 region comprising the amino acidsequence of SEQ ID NO: 27; and a CDR3 region comprising the amino acidsequence of SEQ ID NO:
 28. 6. The monoclonal antibody of claim 1,wherein the VL comprises the amino acid sequence of SEQ ID NOs: 4, 14 or24, and wherein the VH comprises the amino acid sequence of SEQ ID NOs:9, 19 or
 29. 7. The monoclonal antibody of claim 1, wherein the VLcomprises the amino acid sequence of SEQ ID NOs: 4 or 14, and whereinthe VH comprises the amino acid sequence of SEQ ID NOs: 9 or
 19. 8. Themonoclonal antibody of claim 1, wherein: (a) the VL comprises the aminoacid sequence of SEQ ID NO:4, and the VH comprises SEQ ID NO: 9; (b) theVL comprises the amino acid sequence of SEQ ID NO:14, and the VHcomprises SEQ ID NO: 19; or (c) the VL comprises the amino acid sequenceof SEQ ID NO:24, and the VH comprises SEQ ID NO:
 29. 9. The monoclonalantibody of claim 1, which is humanized.
 10. The monoclonal antibody ofclaim 1, which is labeled.
 11. A pharmaceutical composition comprisingthe monoclonal antibody of claim 1 and at least one pharmaceuticalexcipient.
 12. An isolated polynucleotide comprising a first nucleicacid encoding a light chain variable region (VL) and a second nucleicacid encoding a heavy chain variable region (VH), wherein the firstnucleic acid comprises a first segment encoding the amino acid sequenceof SEQ ID NOs: 1, 11 or 21; a second segment encoding the amino acidsequence of SEQ ID NOs: 2, 12 or 22; and a third segment encoding theamino acid sequence of SEQ ID NOs: 3, 13 or 23, and wherein the secondnucleic acid comprises a first segment encoding the amino acid sequenceof SEQ ID NOs: 6, 16 or 26; a second segment encoding the amino acidsequence of SEQ ID NOs: 7, 17 or 27; and a third segment encoding theamino acid sequence of SEQ ID NOs: 8, 18 or
 28. 13. The polynucleotideof claim 12, comprising at least one of the nucleic acid sequences ofSEQ ID NOs: 5, 10, 15, 20, 25 or
 30. 14. The polynucleotide of claim 12,comprising: (a) the nucleic acid sequences of SEQ ID NOs: 5 and 10; (b)the nucleic acid sequences of SEQ ID NOs: 15 and 20; or (c) the nucleicacid sequences of SEQ ID NOs: 25 and
 30. 15. A method of treating asynucleopathic disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the isolated monoclonal antibody of claim
 1. 16. The method ofclaim 15, wherein the synucleopathic disease is at least one from thegroup consisting of Parkinson's disease, Parkinson's disease withdementia, Dementia with Lewy bodies, Alzheimer's disease, Down'ssyndrome, multiple-system atrophy, prion diseases, and other α-Synrelated neurodegenerative disorders.
 17. The method of claim 15, whereinthe antibody is provided to the subject as a pharmaceutical composition.18. The method of claim 15, wherein the antibody is administeredparenterally to the subject.
 19. A method of detecting a synucleopathicdisease in a subject, the method comprising administering to the subjecta labeled isolated monoclonal antibody of claim 1, and detectingpresence or absence of a complex of the labeled isolated monoclonalantibody with any α-Syn fibrils present in the subject, wherein, if thecomplex is detected, the subject has a synucleopathic disease.
 20. Amethod of detecting α-Syn fibrils in a sample, the method comprisingcontacting the sample with a labeled isolated monoclonal antibody ofclaim 1, and detecting presence or absence of a complex of the labeledisolated monoclonal antibody with any α-Syn fibrils present in thesample, wherein, if the complex is detected, α-Syn fibrils are presentin the sample.